| Assay Method Information | |
| | CD73 Biochemical Assay |
| Description: | Compound serial dilution (1:3) was prepared with Echo 555 liquid handler (Labcyte) into the corresponding wells of a 384-well plate. 25 μL of enzyme working solution [0.2 nM recombinant CD73 protein (purchased from R&D Systems, Inc), 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35 non-ionic detergent] was added to the assay plate and incubated with compounds for 15 minutes at room temperature. After adding 25 μL of AMP working solution (10 μM AMP, 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.01% BSA, 0.01% Brij-35) into the assay plate, the reaction was incubated for 10 min at room temperature. 10 uL of Malachite Green A solution was then added to each well for 10 minutes incubation. 10 uL of Malachite B solution was added to each well and then incubated for another 30 minutes. Finally the absorbance value was read on the Envision plate reader (PerkinElmer) at 620 nM. Calculation percentage inhibition by using equation {% inhibition=100×[I−(X−MIN)/(MAX−MIN)]} where X equals to the well signal, Max equals signal of DMSO control and MIN equals signal of no cell control |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |